Mitochondrial stress-70 protein markers for colorectal cancer

ABSTRACT

Provided are previously uncharacterised markers of cancers, for example colorectal cancers, and uses of these as diagnostic and prognostic markers of cancers, and in particular colorectal cancers. The markers are SEQ ID NO: 1—hnRNP-K; SEQ ID NO:2—HMG-1; SEQ ID NO:3—proteasome subunit alpha type 1; SEQ ID NO:4—bifunctional purine biosynthesis protein; SEQ ID NO:5—ST11; SEQ ID NO:6—annex in IV; SEQ ID NO:7—60 kDa heat shock protein; SEQ ID NO:8—T complex protein 1 beta subunit; SEQ ID NO:9—T complex protein 1 epsilon subunit; SEQ ID NO: 10—mortalin; and SEQ ID NO: 11—TER-ATPase. The invention further provides related methods and materials for the use of the markers in therapeutic intervention in colorectal and other cancers e.g. to specifically target neoplastic cells without causing significant toxicity in healthy tissues, and to provide methods for the evaluation of the ability of candidate therapeutic compounds to modulate the biological activity of cancerous cells from the colon, rectum and other tissues.

BACKGROUND OF THE INVENTION

Cancer remains one of the leading causes of death in the Western world.Clinically, the treatment of human cancer currently involves the use ofa broad variety of medical approaches, including surgery, radiationtherapy and chemotherapeutic drug therapy (see, for example, the OxfordTextbook of Oncology, Souhami R L, Tannock I, Hohenberger P, and HoriotJ-C (ed.s), 2nd edition, New York, N.Y., Oxford University Press, 2002).

A diverse group of chemotherapeutic agents are used in the treatment ofhuman cancer, including the taxanes paclitaxel and docetaxel, thetopoisomerase inhibitors etoposide, topotecan and irinotecan, theantimetabolites methotrexate, 5-fluorouracil, 5-fluorodeoxyuridine,6-mercaptopurine, 6-thioguanine, cytosine arabinoside, 5-aza-cytidineand hydroxyurea; the alkylating agents cyclophosphamide, melphalan,busulfan, CCNU, MeCCNU, BCNU, streptozotocin, chlorambucil,bis-diamminedichloroplatinum, azetidinylbenzoquinone; the plantalkaloids vincristine, vinblastine, vindesine, and VM-26; theantibiotics actinomycin-D, doxorubicin, daunorubicin, mithramycin,mitomycin C and bleomycin; and miscellaneous agents such as dacarbazine,mAMSA and mitoxantrone. However, some neoplastic cells developresistance to specific chemotherapeutic agents or even to multiplechemotherapeutic agents, and some tumours are intrinsically resistant tocertain chemotherapeutic agents. Such drug resistance or multiple drugresistance can theoretically arise from expression of genes that conferresistance to the agent, or from lack of expression of genes that makethe cells sensitive to a particular anticancer drug.

It is well established that certain pathological conditions, includingcancer, are characterized by the abnormal expression of certainmolecules, and these molecules thus serve as “markers” for a particularpathological condition.

Apart from their use as diagnostic “targets”, i.e. abnormal componentsthat can be identified to diagnose the pathological condition, themolecules serve as reagents which can be used to generate diagnosticand/or therapeutic agents. An example of this, which is not intended tobe limiting, is the use of markers of cancer to produce antibodiesspecific to a particular marker. A further non-limiting example is theuse of a peptide which complexes with an MHC molecule, to generatecytolytic T cells against cells expressing the marker.

One particular cancer target of interest is colorectal cancer.Colorectal cancers are the third most common malignancies in the world,and amongst men in the European Union it is the second most common causeof cancer death after lung cancer. Although more than 90% of cases arecurable when diagnosed at an early stage in development, the majority ofpatients with colorectal cancer present clinically when the tumour is atan advanced, metastatic stage. Consequently, the disease kills around98,500 people every year in the EU (where less than 50% of patientssurvive 5 years after an initial diagnosis of colorectal cancer) and anestimated 437,000 people per annum worldwide. This problem of latediagnosis is compounded by the resistance of some patients' tumours tocurrently available chemotherapy; leading to a failure to respond totreatment. Such patients require earlier detection and more successfultreatment of their illness, and to this end it is desirable to identifyproteins whose expression is associated with cancerous cells, which mayserve as diagnostic markers, prognostic indicators and therapeutictargets.

Colorectal cancer is a consequence of pathologic transformation ofnormal cells of the colonic epithelium to an invasive cancer, and mayresult from inherited mutation, spontaneous mutation or exposure tocarcinogens in the bowel contents. The majority of cancers of thecolorectum are adenocarcinomas (Jass & Morson, J. Clin. Pathol. 40:1016-23, 1987), but questions remain concerning the true origins ofcolorectal carcinomas. Such carcinomas may arise both from withinexisting benign neoplasms (“adenomas”), in what has been termed theadenoma to carcinoma sequence (Muto et al, Cancer 30: 2251-70, 1975),but the majority of adenomas do not appear to progress to carcinoma andindeed may even regress (Knoemschild, Surg. Forum XIV: 137-8, 1963).Alternatively carcinomas may arise de novo from areas of generaliseddysplasia without an adenomatous stage. Clinical evidence supports theidentification of environment, diet, age and sex as risk factors forcolorectal cancer, but the lack of confirmed involvement of thesefactors in all cases suggests an underlying genetic basis for colorectaltumour formation. Several genetic alterations have been implicated indevelopment of colorectal cancer, including mutations intumour-suppressor genes, proto-oncogenes and DNA repair genes (reviewedby Robbins & Itzkowitz, Med Clin North Am 86:1467-95, 2002; Fearnhead etal, Br Med Bull 64: 27-43, 2002). For example, WO 0077252 identifies theBarx2 gene as a candidate tumour suppressor implicated in ovarian andcolorectal cancer.

One of the earliest detectable events, which may be the initiating eventin colorectal tumourigenesis, is inactivating mutation of both allelesof the adenomatous polyposis coli (APC) tumour suppressor gene. Otherimplicated genes include MCC, p53, DCC (deleted in colorectalcarcinoma), and genes in the TGF-beta signalling pathway. Tumourspecific patterns of expression have also been demonstrated for a numberof proteins in colorectal tissues, and these proteins are undergoingevaluation as diagnostic and therapeutic targets. One such protein iscarcinoembryonic antigen (CEA), which is detectable in the majority ofcolorectal cancers but not in normal tissues (reviewed by Hammarstrom,Semin Cancer Biol 9: 67-81, 1999). CEA is immunologically detectable inthe serum of colorectal cancer patients, and detection of CEA mRNA byRT-PCR can identify lymph node micrometastases, which are a prognosticindicator of a reduced chance of survival in colorectal cancers (Lieferset al, New England J. of Med. 339: 223-8, 1998). Another promisingmarker for colorectal cancer is minichromosome maintenance protein 2(MCM2), which is being developed as a target for diagnosis from stoolsamples (Davies et al, Lancet 359: 1917-9, 2002).

At the present time, none of the protein markers under investigation arein routine clinical use, and further targets for diagnosis, prognosisand treatment are desirable. The current routine diagnostic test forcolorectal cancer is the FOBT (Faecal Occult Blood Test), which islacking in sensitivity and specificity. Evaluation of the effectivenessof this test indicates that it may fail to detect as many as 76% ofsuspicious growths (Lieberman et al, N Engl J Med; 345: 555-60, 2001).It also results in a large number of false positives and these patientsrequire to undergo the unpleasant, invasive procedure of colonoscopy.Even when administered together these two procedures have been found tomiss 24% of tumours and precancerous polyps. Currently the bestcandidates for new diagnostic tests are based on DNA analysis, but thesehave at best a 50% detection rate.

It will be appreciated from the forgoing that the provision of novelspecific, reliable markers that are differentially expressed in normaland transformed tissues (such as colorectal tissue) would provide auseful contribution to the art. Such markers could be used inter alia inthe diagnosis of cancers such as colorectal cancer, the prediction ofthe onset of cancers such as colorectal cancer, or the treatment ofcancers such as colorectal cancer.

SUMMARY OF THE INVENTION

The present inventors have used specific proteomics approaches toidentify proteins that are expressed in cancer cells but not in normaltissues. The target proteins of the present invention are listed inTable 2 and discussed in Example 2 herein.

Each marker has been identified by up-regulation of expression incolorectal tumour samples, an observation not previously made in thistissue type for any of these markers.

Previously, proteome studies have proven to be of limited success inidentifying such markers. W09842736 and W09843091 do disclose certaindifferentially-expressed protein markers identified by proteomicanalysis of clinical samples following laborious processing intended toenrich tumour epithelial cells by removing stromal cells and connectivetissue contaminants. However, more recently, a proteomic comparison ofmurine normal and neoplastic colon tissues identified no statisticallysignificant differences in protein expression patterns (Cole et al,Electrophoresis 21: 1772-81, 2000).

After the presently claimed priority date, the following studies werepublished relating to some of the markers disclosed herein: Kuniyasu H,Chihara Y, Kondo H, Ohmori H, Ukai R (2003) Amphoterin induction inprostatic stromal cells by androgen deprivation is associated withmetastatic prostate cancer. Oncol Rep. 10(6): 1863-8; Cappello F,Bellafiore M, Palma A, David S, Marciano V, Bartolotta T, Sciume C,Modica G, Farina F, Zummo G, Bucchieri F. (2003) 60 KDa chaperonin(HSP60) is over-expressed during colorectal carcinogenesis. EuropeanJournal of Histochemistry 47 (2): 105-10; Yamamoto S, Tomita Y, HoshidaY, Sakon M, Kameyama M, Imaoka S, Sekimoto M, Nakamori S, Monden M,Aozasa K. (2004) Expression of valosin-containing protein in colorectalcarcinomas as a predictor for disease recurrence and prognosis. ClinicalCancer Research 10(2): 651-7.

Other publications concerning the present markers are discussed inExample 2 herein.

Accordingly, the present invention describes the use of the targetproteins listed in Table 2 (which may be referred to hereinafter as “thetarget proteins of the present invention”) as markers of cancer, andprovides methods for their use in such applications.

As discussed in detail below, the target proteins of the presentinvention are of particular use inter alia as diagnostic and prognosticmarkers of cancers, and in particular colorectal cancers. As with knownmarkers, they may be used for example to assist diagnosing the presenceof cancer at an early stage in the progression of the disease andpredicting the likelihood of clinically successful outcome, particularlywith regard to the sensitivity or resistance of a particular patient'stumour to a chemotherapeutic agent or combinations of chemotherapeuticagents. Furthermore these targets can be used for therapeuticintervention in colorectal and other cancers e.g. to specifically targetneoplastic cells without causing significant toxicity in healthytissues, and to provide methods for the evaluation of the ability ofcandidate therapeutic compounds to modulate the biological activity ofcancerous cells from the colon, rectum and other tissues.

Thus the present invention relates to the diagnosis and treatment ofcancer, and specifically to the discrimination of neoplastic cells fromnormal cells on the basis of over-expression of specific tumour antigensand the targeting of treatment through exploitation of the differentialexpression of these antigens within neoplastic cells. The inventionspecifically relates to the detection of one or more proteins (“targetproteins”) that are over-expressed in neoplastic cells compared with theexpression in pathologically normal cells (see Table 2). Furthermore theinvention provides evidence for up-regulation of expression of thistarget in tumour cells where this has not previously been reported.Accordingly, this protein, as well as nucleic acid sequences encodingthis protein, or sequences complementary thereto, can be used as acancer marker useful in diagnosing or predicting the onset of a cancersuch as colorectal cancer, monitoring the efficacy of a cancer therapyand/or as a target of such a therapy.

The invention in particular relates to the discrimination of neoplasticcells from normal cells on the basis of the over-expression of a targetprotein of the present invention, or the gene that encodes this protein.To enable this identification, the invention provides a pattern ofexpression of a specific protein, the expression of which is increasedin neoplastic cells in comparison to normal cells. The inventionprovides a variety of methods for detecting this protein and theexpression pattern of this protein and using this information for thediagnosis and treatment of cancer.

Furthermore, it is contemplated that the skilled artisan may producenovel therapeutics for treating colorectal cancer which include, forexample: antibodies which can be administered to an individual that bindto and reduce or eliminate the biological activity of the target proteinin vivo; nucleic acid or peptidyl nucleic acid sequences which hybridizewith genes or gene transcripts encoding the target proteins thereby toreduce expression of the target proteins in vivo; or small molecules,for example, organic molecules which interact with the target proteinsor other cellular moieties, for example, receptors for the targetprotein, thereby to reduce or eliminate the biological activity of thetarget protein.

The invention therefore further provides methods for targeting oftherapeutic treatments for cancers by directing treatment against thisover-expressed protein. Methods for achieving this targeting mayinclude, but are not limited to;

-   -   (i) conjugation of therapeutic drugs to a moiety such as an        immunoglobulin or aptamer that specifically recognises the        molecular structure of the target protein,    -   (ii) exposure of the host immune system to the target protein or        fragments thereof by immunisation using proteins, polypeptides,        expression vectors or DNA vaccine constructs in order to direct        the host immune system against neoplastic cells in which the        target protein is over-expressed,    -   (iii) modification of the biological activity of the target        protein by small molecule ligands,    -   (iv) exploitation of the biological activity of the target        protein to activate prodrugs,    -   (v) modulation of the expression of the target protein in cells        by methods such as antisense gene silencing, use of small        interfering RNA molecules, or the targeting of regulatory        elements in the gene encoding the target protein or regulatory        proteins that bind to these elements,    -   (vi) specific modulation of the physical interaction of the        target protein with other components of the cell, with for        example a small molecule ligand or an immunoglobulin, in order        to exert a therapeutic benefit

The present invention thereby provides a wide range of novel methods forthe diagnosis, prognosis and treatment of cancers, including colorectalcancer, on the basis of the differential expression of the targetprotein. These and other numerous additional aspects and advantages ofthe invention will become apparent to the skilled artisan uponconsideration of the following detailed description of the invention.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

In the studies disclosed herein, proteomic analysis was applied tocolorectal samples from a clinical tissue bank into which have beencollected both tumour and pathologically normal (disease-free) tissuesfrom each individual donor. Using a process whereby proteins arerecovered from frozen sections of donor tissues selected from a bank offresh frozen tissues on the basis of optimal tumour histology andcellularity, it was possible to derive the protein expression profilesof carefully selected sets of normal colon tissue and advanced (Duke's Cstage) colorectal carcinomas selected from 16 patients. Comparison ofthe protein expression “fingerprints” of the tumour and normal tissuesets revealed differences that arise as a result of the disease process.Table 1 provides details of proteins identified by these means whoseup-regulation in colorectal tumour tissues has previously been reported.Table 2 provides details of proteins identified by these means whoseup-regulation in colorectal tumour tissues has not been previouslydemonstrated and thereby provides the basis of the present invention.

The objective of the present study was to identify new targets forcancer diagnosis and therapy. Accordingly, a first aspect of the presentinvention provides a method for the identification of cancer cells,which method comprises determining the expression of the target proteinof the invention in a sample of tissue from a first individual andcomparing the pattern of expression observed with the pattern ofexpression of the same protein in a second clinically normal tissuesample from the same individual or a second healthy individual, with thepresence of tumour cells in the sample from the first individualindicated by a difference in the expression patterns observed.

More specifically, the invention provides a diagnostic assay forcharacterising tumours and neoplastic cells, particularly humanneoplastic cells, by the differential expression of the target proteinwhereby the neoplastic phenotype is associated with, identified by andcan be diagnosed on the basis thereof. This diagnostic assay comprisesdetecting, qualitatively or preferably quantitatively, the expressionlevel of the target protein and making a diagnosis of cancer on thebasis of this expression level.

In this context, “determining the expression” means qualitative and/orquantitative determinations, of the presence of the target protein ofthe invention including measuring an amount of biological activity ofthe target protein in terms of units of activity or units activity perunit time, and so forth.

As used herein, the term “expression” generally refers to the cellularprocesses by which a polypeptide is produced from RNA.

As used herein, the term “cancer” encompasses cancers in all forms,including polyps, neoplastic cells and preneoplastic cells and includessarcomas and carcinomas. Exemplary sarcomas and carcinomas include, butare not limited to, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumour, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumour, cervical cancer,testicular tumour, lung carcinoma (including small cell lung carcinomaand non-small cell lung carcinoma), bladder carcinoma, epithelialcarcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma ;leukaemias, e. g., acute lymphocytic leukaemia and acute myelocyticleukaemia (myeloblastic, promyelocytic, myelomonocytic, monocytic anderythroleukaemia); chronic leukaemia (chronic myelocytic (granulocytic)leukaemia and chronic lymphocytic leukaemia); and polycythemia vera,lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiplemyeloma, Waldenstrbom's macroglobulinemia, and heavy chain disease.

In a preferred embodiment of the present invention, this method may beapplied to diagnosis of colorectal cancer. The terms “colon cancer”,“rectal cancer”, and “colorectal cancer” are used interchangeablyherein.

Species variants are also encompassed by this invention where thepatient is a non-human mammal, as are allelic or other variants of theproteins described in Table 2, and any reference to the proteins in thattable will be understood to embrace, alleles, homologues or othernaturally occurring variants.

Thus included within the definition of the target protein of theinvention are amino acid variants of the naturally occurring sequence asprovided in any of SEQ ID NOs: 1-11. Preferably, variant sequences areat least 75% homologous to the wild-type sequence, more preferably atleast 80% homologous, even more preferably at least 855 homologous, yetmore preferably at least 90% homologous or most preferably at least 955homologous to at least a portion of the reference sequence supplied (SEQID NOs: 1-11). In some embodiments the homology will be as high as 94 to96 or 98%. Homology in this context means sequence similarity oridentity, with identity being preferred. To determine whether acandidate peptide region has the requisite percentage similarity oridentity to a reference polypeptide or peptide oligomer, the candidateamino acid sequence and the reference amino acid sequence are firstaligned using a standard computer programme such as are commerciallyavailable and widely used by those skilled in the art. In a preferredembodiment the NCBI BLAST method is used www.ncbi.nlm.nih.gov/BLAST/).Once the two sequences have been aligned, a percent similarity score maybe calculated. In all instances, variants of the naturally-occurringsequence, as detailed in SEQ ID NO: 1-11 herein, must be confirmed fortheir function as marker proteins. Specifically, their presence orabsence in a particular form or in a particular biological compartmentmust be indicative of the presence or absence of cancer in anindividual. This routine experimentation can be carried out by usingstandard methods known in the art in the light of the disclosure herein.

In one aspect of the present invention, the target protein can bedetected using a binding moiety capable of specifically binding themarker protein. By way of example, the binding moiety may comprise amember of a ligand-receptor pair, i.e. a pair of molecules capable ofhaving a specific binding interaction. The binding moiety may comprise,for example, a member of a specific binding pair, such asantibody-antigen, enzyme-substrate, nucleic acid-nucleic acid,protein-nucleic acid, protein-protein, or other specific binding pairknown in the art. Binding proteins may be designed which have enhancedaffinity for the target protein of the invention. Optionally, thebinding moiety may be linked with a detectable label, such as anenzymatic, fluorescent, radioactive, phosphorescent, coloured particlelabel or spin label. The labelled complex may be detected, for example,visually or with the aid of a spectrophotometer or other detector.

A preferred embodiment of the present invention involves the use of arecognition agent, for example an antibody recognising the targetprotein of the invention, to contact a sample of tissues, cells, bloodor body product, or samples derived therefrom, and screening for apositive response. The positive response may for example be indicated byan agglutination reaction or by a visualisable change such as a colourchange or fluorescence, e.g. immunostaining,.or by a quantitative methodsuch as in use of radio-immunological methods or enzyme-linked antibodymethods.

The method therefore typically includes the steps of (a) obtaining froma patient a tissue sample to be tested for the presence of cancer cells;(b) producing a prepared sample in a sample preparation process; (c)contacting the prepared sample with a recognition agent, such as anantibody, that reacts with the target protein of the invention; and (d)detecting binding of the recognition agent to the target protein, ifpresent, in the prepared sample. The human tissue sample can be from thecolon or any other tissue in which tumour-specific expression of theappropriate protein can be demonstrated. The sample may further comprisesections cut from patient tissues or it may contain whole cells or itmay be, for example, a body fluid sample selected from the groupconsisting of: blood; serum; plasma; fecal matter; urine; vaginalsecretion; breast exudate; spinal fluid; saliva; ascitic fluid;peritoneal fluid; sputum; and colorectal exudate, or an effusion, wherethe sample may contain cells, or may contain shed antigen. A preferredsample preparation process includes tissue fixation and production of athin section. The thin section can then be subjected toimmunohistochemical analysis to detect binding of the recognition agentto the target protein. Preferably, the immunohistochemical analysisincludes a conjugated enzyme labelling technique. A preferred thinsection preparation method includes formalin fixation and wax embedding.Alternative sample preparation processes include tissue homogenisation.When sample preparation includes tissue homogenisation, a preferredmethod for detecting binding of the antibody to the target protein isWestern blot analysis.

Alternatively, an immunoassay can be used to detect binding of theantibody to the target protein. Examples of immunoassays are antibodycapture assays, two-antibody sandwich assays, and antigen captureassays. In a sandwich immunoassay, two antibodies capable of binding themarker protein generally are used, e.g. one immobilised onto a solidsupport, and one free in solution and labelled with a detectablechemical compound. Examples of chemical labels that may be used for thesecond antibody include radioisotopes, fluorescent compounds, spinlabels, coloured particles such as colloidal gold and coloured latex,and enzymes or other molecules that generate coloured orelectrochemically active products when exposed to a reactant or enzymesubstrate. When a sample containing the marker protein is placed in thissystem, the marker protein binds to both the immobilised antibody andthe labelled antibody, to form a “sandwich” immune complex on thesupport's surface. The complexed protein is detected by washing awaynon-bound sample components and excess labelled antibody, and measuringthe amount of labelled antibody complexed to protein on the support'ssurface. Alternatively, the antibody free in solution, which can belabelled with a chemical moiety, for example, a hapten, may be detectedby a third antibody labelled with a detectable moiety which binds thefree antibody or, for example, the hapten coupled thereto. Preferably,the immunoassay is a solid support-based immunoassay. Alternatively, theimmunoassay may be one of the immunoprecipitation techniques known inthe art, such as, for example, a nephelometric immunoassay or aturbidimetric immunoassay. When Western blot analysis or an immunoassayis used, preferably it includes a conjugated enzyme labelling technique.

Although the recognition agent will conveniently be an antibody, otherrecognition agents are known or may become available, and can be used inthe present invention. For example, antigen binding domain fragments ofantibodies, such as Fab fragments, can be used. Also, so-called RNAaptamers may be used. Therefore, unless the context specificallyindicates otherwise, the term “antibody” as used herein is intended toinclude other recognition agents. Where antibodies are used, they may bepolyclonal or monoclonal. Optionally, the antibody can be produced by amethod such that it recognizes a preselected epitope from the targetprotein of the invention.

The isolated target protein of the invention may be used for thedevelopment of diagnostic and other tissue evaluation kits and assays tomonitor the level of the proteins in a tissue or fluid sample. Forexample, the kit may include antibodies or other specific bindingmoieties which bind specifically to the target protein which permit thepresence and/or concentration of the colorectal cancer-associatedproteins to be detected and/or quantified in a tissue or fluid sample.Accordingly, the invention further provides for the production ofsuitable kits for detecting the target protein, which may for exampleinclude a receptacle or other means for receiving a sample to beevaluated, and a means for detecting the presence and/or quantity in thesample of the target protein of the invention and optionallyinstructions for performing such an assay.

In a further aspect of the present invention is provided herein a methodof evaluating the effect of a candidate therapeutic drug for thetreatment of cancer, said method comprising administering said drug to apatient, removing a cell sample from said patient; and determining theexpression profile of the target protein of the invention in said cellsample. This method may further comprise comparing said expressionprofile to an expression profile of a healthy individual. In a preferredembodiment, said patient is receiving treatment for colorectal cancerand said cell sample is derived from tissues of the colon and/or rectum.In a further preferred embodiment the present invention further providesa method for determine the efficacy of a therapeutic regime at one ormore timepoints, said method comprising determining a baseline value forthe expression of the protein being tested in a given individual withina given tissue such as a tumour, administering a given therapeutic drug,and then redetermining expression levels of the protein within thatgiven tissue at one or more instances thereafter, observing changes inprotein levels as an indication of the efficacy of the therapeuticregime.

In a further aspect of the present invention the target protein of theinvention provides a mechanism for the selective targeting ofanti-cancer drugs based on metabolism by the target protein withintumours. The present invention therefore provides for the design of, orscreening for, drugs that undergo specific metabolism in tumoursmediated by the target protein of the invention, whereby this metabolismconverts a non-toxic moiety into a toxic one, which kills or inhibitsthe tumour or makes it more susceptible to other agents. In a furtherpreferred embodiment of the present invention, a method of treatingcolorectal cancer is provided, said method comprising use of a drug thatis specifically metabolised to an active form by contact with the targetprotein of the invention.

A further aspect of the invention provides for the targeting ofcytotoxic drugs or other therapeutic agents, or the targeting of imagingagents, by virtue of their recognition of epitopes derived from thetarget protein of the invention on the surface of a tumour cell, whetheras part of the complete target protein itself or in some degraded formsuch as in the presentation on the surface of a cell bound to a MHCprotein.

A further embodiment of the present invention is the development oftherapies for treatment of conditions which are characterized byover-expression of the target protein of the invention viaimmunotherapeutic approaches. More specifically, the invention providesmethods for stimulation of the immune system of cancer patients, forexample by activating cytotoxic or helper T-cells which recogniseepitopes derived from the protein of the invention so as to implement acell-mediated or humoral immune response against the tumour. By way ofexample, the activation of the immune system can be achieved byimmunisation with sequences derived from the target protein of theinvention in an amount sufficient to provoke or augment an immuneresponse. By way of further example, which is specifically not intendedto limit the scope of the invention, these may be administered as nakedpeptides, as peptides conjugated or encapsulated in one or moreadditional molecules (e.g. liposomes) such that a pharmacologicalparameter (e.g. tissue permeability, resistance to endogenousproteolysis, circulating half-life etc) is improved, or in a suitableexpression vector which causes the expression of the sequences at anappropriate site within the body to provoke an immune response. Theproteins or peptides may be combined with one or more of the knownimmune adjuvants, such as saponins, GM-CSF, interleukins, and so forth.Peptides that are too small to generate a sufficient immune responsewhen administered alone can be coupled to one or more of the variousconjugates used to stimulate such responses which are well known in theart. Furthermore, peptides which form non-covalent complexes with MHCmolecules within cells of the host immune system may be used to elicitproliferation of cytolytic T cells against any such complexes in thesubject. Such peptides may be administered endogenously or may beadministered to isolated T-cells ex-vivo and then reperfused into thesubject being treated. Alternatively, the generation of a host immuneresponse can be accomplished by administration of cells, preferablyrendered non-proliferative by standard methods, which present relevant Tcell or B cell epitopes to trigger the required response.

Because up-regulation of expression of the target protein of theinvention is associated with tumour cells, it is likely that theseproteins in some way contribute to the process of tumourigenesis or thepersistence of tumour cells. Consequently, the present inventionprovides for the reduction of the expression level of the target proteinin tumour cells, for example by the use of suicide inhibitors or byusing antisense RNA methods to decrease the synthesis of the protein.Similarly, this reduction in expression levels could also be achieved bydown-regulation of the corresponding gene promoter. A preferred methodcomprises the step of administering to a patient diagnosed as havingcancer, such as colorectal cancer, a therapeutically-effective amount ofa compound which reduces in vivo the expression of the target protein.In a preferred embodiment, the compound is a polynucleotide, forexample, an anti-sense nucleic acid sequence or a peptidyl nucleic acid(PNA), more preferably from 10 to 100 nucleotides in length, capable ofbinding to and reducing the expression (for example, transcription ortranslation) of a nucleic acid encoding at least a portion of the targetprotein of the invention. After administration, the anti-sense nucleicacid sequence or the anti-sense PNA molecule binds to the nucleic acidsequences encoding, at least in part, the target protein thereby toreduce in vivo expression of the target protein. By way of furtherexample, constructs of the present invention capable of reducingexpression of the target protein can be administered to the subjecteither as a naked polynucleotide or formulated with a carrier, such as aliposome, to facilitate incorporation into a cell. Such constructs canalso be incorporated into appropriate vaccines, such as in viral vectors(e.g. vaccinia), bacterial constructs, such as variants of the wellknown BCG vaccine, and so forth.

A particularly useful therapeutic embodiment of the present inventionprovides an oligonucleotide or peptidyl nucleic acid sequencecomplementary and capable of hybridizing under physiological conditionsto part, or all, of the gene encoding the target protein or to part, orall, of the transcript encoding the target protein thereby to reduce orinhibit transcription and/or translation of the target protein gene.

Anti-sense oligonucleotides have been used extensively to inhibit geneexpression in normal and abnormal cells. For a recent review, seePhillips, ed., Antisense Technology, in Methods in Enzymology, vols.313-314, Academic Press; Hartmann, ed., 1999. In addition, the synthesisand use of peptidyl nucleic acids as anti-sense-based therapeutics aredescribed in PCT publications PCT/EP92/01219, PCT/US92/1092, andPCT/US94/013523. Accordingly, the anti-sense-based therapeutics may beused as part of chemotherapy, either alone or in combination with othertherapies.

Double stranded RNA (dsRNA) has been found to be even more effective ingene silencing than both sense or antisense strands alone (Fire A. et alNature, Vol 391, (1998)). dsRNA mediated silencing is gene specific andis often termed RNA interference (RNAi) (See also Fire (1999) TrendsGenet. 15: 358-363, Sharp (2001) Genes Dev. 15: 485-490, Hammond et al.(2001) Nature Rev. Genes 2: 1110-1119 and Tuschl (2001) Chem. Biochem.2: 239-245).

RNA interference is a two step process. First, dsRNA is cleaved withinthe cell to yield short interfering RNAs (siRNAs) of about 21-23ntlength with 5′ terminal phosphate and 3′ short overhangs (˜2nt) ThesiRNAs target the corresponding mRNA sequence specifically fordestruction (Zamore P. D. Nature Structural Biology, 8, 9, 746-750,(2001)

Thus in one embodiment, the invention provides double stranded RNAcomprising a sequence encoding a target protein of the presentinvention, which may for example be a “long” double stranded RNA (whichwill be processed to siRNA, e.g., as described above). These RNAproducts may be synthesised in vitro, e.g., by conventional chemicalsynthesis methods.

RNAi may be also be efficiently induced using chemically synthesizedsiRNA duplexes of the same structure with 3′-overhang ends (Zamore P Det al Cell, 101, 25-33, (2000)). Synthetic siRNA duplexes have beenshown to specifically suppress expression of endogenous and heterologousgenes in a wide range of mammalian cell lines (Elbashir S M. et al.Nature, 411, 494-498, (2001)). Thus siRNA duplexes containing between 20and 25 bps, more preferably between 21 and 23 bps, of the sequenceencoding a target protein of the present invention form one aspect ofthe invention e.g. as produced synthetically, optionally in protectedform to prevent degradation. Alternatively siRNA may be produced from avector, in vitro (for recovery and use) or in vivo.

Accordingly, the vector may comprise a nucleic acid sequence encoding atarget protein of the present invention (including a nucleic acidsequence encoding a variant or fragment thereof), suitable forintroducing an siRNA into the cell in any of the ways known in the art,for example, as described in any of references cited herein, whichreferences are specifically incorporated herein by reference.

In one embodiment, the vector may comprise a nucleic acid sequenceaccording to the invention in both the sense and antisense orientation,such that when expressed as RNA the sense and antisense sections willassociate to form a double stranded RNA. This may for example be a longdouble stranded RNA (e.g., more than 23nts) which may be processed inthe cell to produce siRNAs (see for example Myers (2003) NatureBiotechnology 21:324-328).

Alternatively, the double stranded RNA may directly encode the sequenceswhich form the siRNA duplex, as described above. In another embodiment,the sense and antisense sequences are provided on different vectors.

These vectors and RNA products may be useful for example to inhibit denovo production of the protein of the present invention in a cell. Theymay be used analogously to the expression vectors in the variousembodiments of the invention discussed herein.

In particular there is provided double-stranded RNA which comprises anRNA sequence encoding a target protein of the present invention or afragment thereof, which may be an siRNA duplex consisting of between 20and 25 bps. Also provided are vectors encoding said dsRNA or siRNAduplexes. Also provided are methods of producing said siRNA duplexescomprising introducing such vectors into a host cell and causing orallowing transcription from the vector in the cell. Separate vectors mayencode: (i) the sense sequence of the siRNA duplex, and (ii) theanti-sense sequence of the siRNA duplex.

An additional DNA based therapeutic approach provided by the presentinvention is the use of a vector which comprises one or more nucleotidesequences, preferably a plurality of these, each of which encodes animmunoreactive peptide derived from the target protein of the invention.Alternatively, a further method of the invention involves combining oneor more of these nucleotide sequences encoding peptides derived from thetarget protein of the invention in combination with nucleotide sequencesencoding peptides derived from other tumour markers known in the art tobe expressed by cancer cells, and encompasses inclusion of suchsequences in all possible variations, such as one from each protein,several from one or more protein and one from each of one or moreadditional proteins, and so forth.

A further aspect of the present invention provides novel methods forscreening for compositions that modulate the expression or biologicalactivity of the target protein of the invention. As used herein, theterm “biological activity” means any observable effect resulting frominteraction between the target protein and a ligand or binding partner.Representative, but non-limiting, examples of biological activity in thecontext of the present invention include association of the targetprotein of the invention with a ligand, such as any of those shown inTable 4.

The term “biological activity” also encompasses both the inhibition andthe induction of the expression of the target protein of the invention.Further, the term “biological activity” encompasses any and all effectsresulting from the binding of a ligand or other in vivo binding partnerby a polypeptide derivative of the protein of the invention. In oneembodiment, a method of screening drug candidates comprises providing acell that expresses the target protein of the invention, adding acandidate therapeutic compound to said cell and determining the effectof said compound on the expression or biological activity of saidprotein. In a further embodiment, the method of screening candidatetherapeutic compounds includes comparing the level of expression orbiological activity of the protein in the absence of said candidatetherapeutic compound to the level of expression or biological activityin the presence of said candidate therapeutic compound. Where saidcandidate therapeutic compound is present its concentration may bevaried, and said comparison of expression level or biological activitymay occur after addition or removal of the candidate therapeuticcompound. The expression level or biological activity of said targetprotein may show an increase or decrease in response to treatment withthe candidate therapeutic compound.

Candidate therapeutic molecules of the present invention may include, byway of example, peptides produced by expression of an appropriatenucleic acid sequence in a host cell or using synthetic organicchemistries, or non-peptide small molecules produced using conventionalsynthetic organic chemistries well known in the art. Screening assaysmay be automated in order to facilitate the screening of a large numberof small molecules at the same time.

As used herein, the terms “candidate therapeutic compound” refers to asubstance that is believed to interact with the target protein of theinvention (or a fragment thereof), and which can be subsequentlyevaluated for such an interaction. Representative candidate therapeuticcompounds include “xenobiotics”, such as drugs and other therapeuticagents, natural products and extracts, carcinogens and environmentalpollutants, as well as “endobiotics” such as steroids, fatty acids andprostaglandins. Other examples of candidate compounds that can beinvestigated using the methods of the present invention include, but arenot restricted to, agonists and antagonists of the target protein of theinvention, toxins and venoms, viral epitopes, hormones (e. g., opioidpeptides, steroids, etc.), hormone receptors, peptides, enzymes, enzymesubstrates, co-factors, lectins, sugars, oligonucleotides or nucleicacids, oligosaccharides, proteins, small molecules and monoclonalantibodies.

In one preferred embodiment the present invention provides a method ofdrug screening utilising eukaryotic or prokaryotic host cells stablytransformed with recombinant polynucleotides expressing the targetprotein of the invention or a fragment thereof, preferably incompetitive binding assays. Such cells, either in viable or fixed form,can be used for standard binding assays. For example, the assay maymeasure the formation of complexes between a target protein and theagent being tested, or examine the degree to which the formation of acomplex between the target protein or fragment thereof and a knownligand or binding partner is interfered with by the agent being tested.Thus, the present invention provides methods of screening for drugscomprising contacting such an agent with the target protein of theinvention or a fragment thereof or a variant thereof found in a tumourcell and assaying (i) for the presence of a complex between the agentand the target protein, fragment or variant thereof, or (ii) for thepresence of a complex between the target protein, fragment or variantand a ligand or binding partner. In such competitive binding assays thetarget protein or fragment or variant is typically labelled. Free targetprotein, fragment or variant thereof is separated from that present in aprotein: protein complex and the amount of free (i.e. uncomplexed) labelis a measure of the binding of the agent being tested to the targetprotein or its interference with binding of the target protein to aligand or binding partner, respectively.

Alternatively, an assay of the invention may measure the influence ofthe agent being tested on a biological activity of the target protein.Thus, the present invention provides methods of screening for drugscomprising contacting such an agent with the target protein of theinvention or a fragment thereof or a variant thereof found in a tumourcell and assaying for the influence of such an agent on a biologicalactivity of the target protein, by methods well known in the art. Insuch activity assays the biological activity of the target protein,fragment or variant thereof is typically monitored by provision of areporter system. For example, this may involve provision of a natural orsynthetic substrate that generates a detectable signal in proportion tothe degree to which it is acted upon by the biological activity of thetarget molecule.

It is contemplated that, once candidate therapeutic compounds have beenelucidated, rational drug design methodologies well known in the art maybe employed to enhance their efficacy. The goal of rational drug designis to produce structural analogues of biologically active polypeptidesof interest or of small molecules with which they interact (e. g.agonists, antagonists, inhibitors) in order to fashion drugs which are,for example, more active or stable forms of the polypeptide, or which,for example, enhance or interfere with the function of a polypeptide invivo. In one approach, one first determines the three-dimensionalstructure of a protein of interest, such as the target protein of theinvention or, for example, of the target protein in complex with aligand, by x-ray crystallography, by computer modelling or mosttypically, by a combination of approaches. For example, the skilledartisan may use a variety of computer programmes which assist in thedevelopment of quantitative structure activity relationships (QSAR) thatact as a guide in the design of novel, improved candidate therapeuticmolecules. Less often, useful information regarding the structure of apolypeptide may be gained by modelling based on the structure ofhomologous proteins. In addition, peptides can be analysed by alaninescanning (Wells, Methods Enzymol. 202: 390-411, 1991), in which eachamino acid residue of the peptide is sequentially replaced by an alanineresidue, and its effect on the peptide's activity is determined in orderto determine the important regions of the peptide. It is also possibleto design drugs based on a pharmacophore derived from the crystalstructure of a target-specific antibody selected by a functional assay.It is further possible to avoid the use of protein crystallography bygenerating anti-idiotypic antibodies to such a functional,target-specific antibody, which have the same three-dimensionalconformation as the original target protein. These anti-idiotypicantibodies can subsequently be used to identify and isolate peptidesfrom libraries, which themselves act as pharmacophores for further usein rational drug design.

For use as a medicament in vivo, candidate therapeutic compounds soidentified may be combined with a suitable pharmaceutically acceptablecarrier, such as physiological saline or one of the many other usefulcarriers well characterized in the medical art. Such pharmaceuticalcompositions may be provided directly to malignant cells, for example,by direct injection, or may be provided systemically, provided theformulation chosen permits delivery of the therapeutically effectivemolecule to tumour cells containing the target protein of the invention.Suitable dose ranges and cell toxicity levels may be assessed usingstandard dose ranging methodology. Dosages administered may varydepending, for example, on the nature of the malignancy, the age, weightand health of the individual, as well as other factors.

A further aspect of the present invention provides for cells and animalswhich express the target protein of the invention and can be used asmodel systems to study and test for substances which have potential astherapeutic agents.

Such cells may be isolated from individuals with mutations, eithersomatic or germline, in the gene encoding the target protein of theinvention, or can be engineered to express or over-express the targetprotein or a variant thereof, using methods well known in the art. Aftera test substance is applied to the cells, any relevant trait of thecells can be assessed, including by way of example growth, viability,tumourigenicity in nude mice, invasiveness of cells, and growth factordependence, assays for each of which traits are known in the art.

Animals for testing candidate therapeutic agents can be selected aftermutagenesis of whole animals or after treatment of germline cells orzygotes. As discussed in more detail below, by way of example, suchtreatments can include insertion of genes encoding the target protein ofthe invention in wild-type or variant form, typically from a secondanimal species, as well as insertion of disrupted homologous genes.Alternatively, the endogenous target protein gene(s) of the animals maybe disrupted by insertion or deletion mutation or other geneticalterations using conventional techniques that are well known in theart. After test substances have been administered to the animals, thegrowth of tumours can be assessed. If the test substance prevents orsuppresses the growth of tumours, then the test substance is a candidatetherapeutic agent for the treatment of those cancers expressing thetarget protein of the invention, for example of colorectal cancers.These animal models provide an extremely important testing vehicle forpotential therapeutic compounds.

Thus the present invention thus provides a transgenic non-human animal,particularly a rodent, which comprises an inactive copy of the geneencoding a target protein of the present invention.

The invention further provides a method of testing a putativetherapeutic of the invention which comprises administering saidtherapeutic to an animal according to the invention and determining theeffect of the therapeutic.

For the purposes of the present invention, it will be understood thatreference to an inactive copy of the gene encoding a target protein ofthe present invention includes any non-wild-type variant of the genewhich results in knock out or down regulation of the gene, andoptionally in a cancer phenotype. Thus the gene may be deleted in itsentirety, or mutated such that the animal produces a truncated protein,for example by introduction of a stop codon and optionally upstreamcoding sequences into the open reading frame of the gene encoding atarget protein of the present invention. Equally, the open reading framemay be intact and the inactive copy of the gene provided by mutations inpromoter regions.

Generally, inactivation of the gene may be made by targeted homologousrecombination. Techniques for this are known as such in the art.

This may be achieved in a variety of ways. A typical strategy is to usetargeted homologous recombination to replace, modify or delete thewild-type gene in an embryonic stem (ES) cell. A targeting vectorcomprising a modified target gene is introduced into ES cells byelectroporation, lipofection or microinjection. In a few ES cells, thetargeting vector pairs with the cognate chromosomal DNA sequence andtransfers the desired mutation carried by the vector into the genome byhomologous recombination. Screening or enrichment procedures are used toidentify the transfected cells, and a transfected cell is cloned andmaintained as a pure population. Next, the altered ES cells are injectedinto the blastocyst of a preimplantation mouse embryo or alternativelyan aggregation chimera is prepared in which the ES cells are placedbetween two blastocysts which, with the ES cells, merge to form a singlechimeric blastocyst. The chimeric blastocyst is surgically transferredinto the uterus of a foster mother where the development is allowed toprogress to term. The resulting animal will be a chimera of normal anddonor cells. Typically the donor cells will be from an animal with aclearly distinguishable phenotype such as skin colour, so that thechimeric progeny is easily identified. The progeny is then bred and itsdescendants cross-bred, giving rise to heterozygotes and homozygotes forthe targeted mutation. The production of transgenic animals is describedfurther by Capecchi, M, R., 1989, Science 244; 1288-1292; Valancius andSmithies, 1991, Mol. Cell. Biol. 11; 1402-1408; and Hasty et al, 1991,Nature 350; 243-246, the disclosures of which are incorporated herein byreference.

Homologous recombination in gene targeting may be used to replace thewild-type gene encoding a target protein of the present invention with aspecifically defined mutant form (e.g. truncated or containing one ormore substitutions).

The inactive gene may also be one in which its expression may beselectively blocked either permanently or temporarily. Permanentblocking may be achieved by supplying means to delete the gene inresponse to a signal. An example of such a means is the cre-lox systemwhere phage lox sites are provided at either end of the transgene, or atleast between a sufficient portion thereof (e.g. in two exons locatedeither side or one or more introns). Expression of a cre recombinasecauses excision and circularisation of the nuclei acid between the twolox sites. Various lines of transgenic animals, particularly mice, arecurrently available in the art which express cre recombinase in adevelopmentally or tissue restricted manner, see for example Tsien,Cell, Vol.87(7): 1317-1326, (1996) and Betz, Current Biology, Vol.6(10):1307-1316 (1996). These animals may be crossed with lox transgenicanimals of the invention to examine the function of the gene encoding atarget protein of the present invention. An alternative mechanism ofcontrol is to supply a promoter from a tetracycline resistance gene,tet, to the control regions of the target gene locus such that additionof tetracycline to a cell binds to the promoter and blocks expression ofthe gene encoding a target protein of the present invention.Alternatively GAL4, VP16 and other transactivators could be used tomodulate gene expression including that of a transgene containing thegene encoding a target protein of the present invention. Furthermore,the target gene could also be expressed in ectopic sites, that is insites where the gene is not normally expressed in time or space.

Transgenic targeting techniques may also be used to delete the geneencoding a target protein of the present invention. Methods of targetedgene deletion are described by Brenner et al, WO94/21787 (Cell Genesys),the disclosure of which is incorporated herein by reference.

In a further embodiment of the invention, there is provided a non-humananimal which expresses the gene encoding a target protein of the presentinvention at a higher than wild-type level. Preferably this means thatthe gene encoding a target protein of the present invention is expressedat least 120-200% of the level found in wild-type animals of the samespecies, when cells which express the gene are compared. Also, this genecould be expressed in an ectopic location where the target gene is notnormally expressed in time or space. Comparisons may be convenientlydone by northern blotting and quantification of the transcript level.The higher level of expression may be due to the presence of one ormore, for example two or three, additional copies of the target gene orby modification to the gene encoding a target protein of the presentinventions to provide over-expression, for example by introduction of astrong promoter or enhancer in operable linkage with the wild-type gene.The provision of animals with additional copies of genes may be achievedusing the techniques described herein for the provision of “knock-out”animals.

In another aspect, animals are provided in which the gene encoding atarget protein of the present invention is expressed at an ectopiclocation. This means that the gene is expressed in a location or at atime during development which does not occur in a wild-type animal. Forexample, the gene may be linked to a developmentally regulated promotersuch as Wnt-1 and others (Echeland, Y. Et al., Development 120,2213-2224, 1998; Rinkenberger, J. C. et al., Dev. Genet. 21, 6-10, 1997,or a tissue specific promoter such as HoxB (Machonochie, M. K. et al,Genes & Dev 11, 1885-1895, 1997).

Non-human mammalian animals include non-human primates, rodents,rabbits, sheep, cattle, goats, pigs. Rodents include mice, rats, andguinea pigs. Amphibians include frogs. Fish such as zebra fish, may alsobe used. Transgenic non-human mammals of the invention may be used forexperimental purposes in studying cancer, and in the development oftherapies designed to alleviate the symptoms or progression of cancer.By “experimental” it is meant permissible for use in animalexperimentation or testing purposes under prevailing legislationapplicable to the research facility where such experimentation occurs.

Other features of the invention will be clear to the skilled artisan,and need not be repeated here. The terms and expressions employed hereinare used as terms of description and not of limitation; there is nointention in the use of such terms and expressions to exclude anyequivalents of the features shown and described or portions thereof, itbeing recognized that various modifications are possible within thescope of the invention.

The disclosure of all references cited herein, inasmuch as it may beused by those skilled in the art to carry out the invention, is herebyspecifically incorporated herein by cross-reference.

Tables and Sequences

Table 1: differentially expressed proteins identified in the presentstudy and already known to be upregulated in colorectal cancer.

Table 2: protein detectable in colorectal cancer samples but not normalcolon tissue controls.

Table 3: Clinicopathological characteristics of the cases used forproteome analysis. All the cases were Dukes C colorectal cancers.

Sequence Annex I:

Seq ID No 1: wild-type amino acid sequence of hnRNP-K as sourced fromthe public SwissProt protein sequence database (SwissProt primaryaccession number Q07244).

Seq ID No 2: wild-type amino acid sequence of HMG-l as sourced from thepublic SwissProt protein sequence database (SwissProt primary accessionnumber P09429).

Seq ID No 3: wild-type amino acid sequence of proteasome subunit alphatype 1 as sourced from the public SwissProt protein sequence database(SwissProt primary accession number P25786).

Seq ID No 4: wild-type amino acid sequence of bifunctional purinebiosynthesis protein as sourced from the public SwissProt proteinsequence database (SwissProt primary accession number P31939).

Seq ID No 5: wild-type amino acid sequence of STI1 as sourced from thepublic SwissProt protein sequence database (SwissProt primary accessionnumber P31948).

Seq ID No 6: wild-type amino acid sequence of annexin IV as sourced fromthe public SwissProt protein sequence database (SwissProt primaryaccession number P09525).

Seq ID No 7: wild-type amino acid sequence of 60 kDa heat shock proteinas sourced from the public SwissProt protein sequence database(SwissProt primary accession number P10809).

Seq ID No 8: wild-type amino acid sequence of T complex protein 1 betasubunit as sourced from the public SwissProt protein sequence database(SwissProt primary accession number P78371).

Seq ID No 9: wild-type amino acid sequence of T complex protein 1epsilon subunit as sourced from the public SwissProt protein sequencedatabase (SwissProt primary accession number P48643).

Seq ID No 10: wild-type amino acid sequence of mortalin as sourced fromthe public SwissProt protein sequence database (SwissProt primaryaccession number P38646).

Seq ID No 11: wild-type amino acid sequence of TER-ATPase as sourcedfrom the public SwissProt protein sequence database (SwissProt primaryaccession number P55072).

EXAMPLE 1 Identification of Novel Markers

Proteins exhibiting differential expression in clinically resectedcolorectal tumours and normal colon tissues were identified as follows;

Tissue Samples

Proteomic analysis was performed on fresh frozen tissue samples obtainedfrom primary colorectal cancer resections and which had been stored inthe Aberdeen colorectal cancer tissue bank. None of the patients in thisstudy disclosed herein had received chemotherapy or radiotherapy priorto surgery. Representative samples of viable tumour and normalcolorectal mucosa (obtained at a distance of at least 5 cm from tumour)were dissected from colorectal cancer excision specimens within 30minutes of surgical removal, and these dissected samples wereimmediately frozen in liquid nitrogen and stored at −80° C. prior toanalysis. Proteomic analysis was performed in duplicate on 16 matchedpairs of frozen tumour and normal colorectal tissue samples. All casesselected were Dukes C colorectal cancers (see Table 3).

Two-Dimensional Gel Electrophoresis

Lysis buffer was prepared according to our established protocols [LawrieL, Curran S, McLeod H L, Fothergill J E, Murray G I. (2001) Applicationof laser capture microdissection and proteomics in colon cancer.Molecular Pathology 54: 253-258.] and contained urea (42% w/v); thiourea(15% w/v); Chaps[3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate] (4% w/v);N-decanoyl-N-methylglucamine (Mega 10, 1% w/v),1-O-Octyl-β-D-glucopyranoside (OBG, 1% w/v), Triton X-100(polyoxyethylene-p-isooctylphenol) (0.5% v/v); Tris[Tris(hydroxymethyl)aminomethane] (0.5% w/v); DTT (dithiothreitol)(0.8%w/v); IPG 3-10 NL (immobilised pH gradient) buffer (1% v/v),β-mercaptoethanol (1% v/v), tributylphosphine (0.02% v/v). All chemicalswere obtained from Amersham Biosciences, UK, with the exception of OBG(Aldrich, UK) and Mega 10 (Sigma, UK).

Frozen sections (10 μm in thickness) of tumour and normal were cut usinga cryostat and thirty 10 μm sections of normal tissue and thirty 10 μmsections of tumour tissue were solubilised in 350 μl and 500 μl of lysisbuffer respectively [Lawrie L, Curran S, McLeod H L, Fothergill J E,Murray G I. (2001) Application of laser capture microdissection andproteomics in colon cancer. Molecular Pathology 54: 253-258]. Onesection from each sample was stained with haematoxylin and eosin toconfirm the diagnosis of each tumour and normal sample; an assessment oftumour cellularity was also made of the tumour sample. 500 μg of normaland tumour sample were loaded, in duplicate, into Immobiline. Dry stripholders and Immobiline Drystrips, pI 3-10 NL, (Amersham Biosciences)were placed into the strip holders. The strips were incubated overnightat room temperature to allow the strips to absorb the samples. Afterincubation the strips were removed, the strip holders cleaned, and smallpieces of dampened electrode strips were then placed over the electrodesin the strip holders to help absorb excess salt during the 1^(st)dimension focusing stage. The strips were then placed back into thestrip holders and covered with dry strip cover fluid (AmershamBiosciences). The 1^(st) dimension focusing was carried out on anIPGPhor system under the following conditions; 30 min at 20V, 1.5 hr at200V, 1.5 hr gradient to 3500V, 35 hr at 3500V, at 15° C. Aftercompletion of focusing the strips were equilibrated for 30 min inequilibration buffer containing urea (36% w/v); 0.5M Tris-HCl, pH 6.9(20% v/v); 20% SDS (dodecyl sulphate, sodium salt) (20% v/v); DTT (0.4%w/v); glycerol (30% v/v). Strips were equilibrated for a further 30 minin equilibration buffer where DTT was replaced by iodoacetamide (1%w/v). All chemicals were obtained from Amersham Biosciences.

Proteins were separated in the 2^(nd) dimension according to theirmolecular weight on a 7 cm NuPAGE 4-12%, 1 well, Bis-Tris gel(Invitrogen, Paisley, UK). 1st dimension strips were attached to the2^(nd) dimension gel with a 4% low melting point agarose solution(Amersham Biosciences). Normal and tumour samples from the same patientwere run in the same gel tank to account for any differences caused bythe gel running process. Gels were run at a constant 120V until thebromophenol dye front reached the end of the gel.

Proteins were visualised using a Colloidal Blue Staining Kit(Invitrogen, Paisley, UK). Gels were fixed in a solution containingmethanol (50% v/v), acetic acid (10% v/v) for 30 min, then transferredto a staining solution containing methanol (20% v/v), Stainer A (20%v/v), Stainer B (5% v/v) for overnight staining to visualise theproteins. Gels were destained using HPLC-grade water with microwaveheating.

Detection of Differential Protein Expression

Destained gels were immediately photographed to produce a black andwhite image. Gel photographs were scanned to produce a computer imagewhich was then enlarged and printed onto sheets of acetate overlayingthe normal and tumour acetate gel pictures allowed proteins which weredifferentially expressed to be detected. Differentially expressedprotein spots were cut from the gel in preparation for identification bymass spectrometry.

Identification of Proteins from Gel

Individual proteins were identified by peptide mass mapping. Proteinspots were cut from the gel, washed to remove Coomassie stain, reducedwith DTT and alkylated with iodoacetamide then digested with trypsin.Trypsin cleaves proteins (at peptide bonds) after arginine and lysineresidues. This action produced a set of tryptic fragments unique to eachprotein. The resultant tryptic peptides were extracted from the gelpieces under full automation (Pro-Gest Robot, Genomic Solutions). Thetryptic fragments were desalted using micro porous tips (Millipore), anddeposited onto a sample plate along with a matrix chemical(α-cyano-4-hydroxycinnamic acid) under full automation (Pro-MS, GenomicSolutions). The masses of the tryptic fragments were then determined byMatrix Assisted Laser Desorption Ionisation Time of Flight MassSpectrometry (MALDI-TOF MS) using a PerSeptive Biosystems Voyager-DE STRmass spectrometer.

To identify the original protein, the masses of the tryptic peptideswere entered into the MS-Fit database-searching program.Database-searching programs attempt to match the experimentally obtainedmasses of tryptic peptides with the theoretically calculated masses oftryptic peptides derived from all proteins within a database. Thedatabase search was restricted to search only for human proteins, norestriction was placed on either the molecular weight or the isoelectricpoint of the protein. To be confident that the correct protein wasidentified, a clear difference in statistical score between the proteinsranked first and second in the results list had to be obtained.

The study disclosed herein identified as differentially-expressed anumber of proteins that have previously been reported as up-regulated incolorectal tumours, thereby validating our experimental methodology andsupporting our novel findings. These are shown in Table 1.

Increased expression of both calgranulin A (calcium binding proteinS100A8, SwissProt Accession Number P05109) and calgranulin B (calciumbinding protein S100A9, SwissProt Accession Number P06702) has beenpreviously reported in colorectal tissues (Stulik J. et al,Electrophoresis 20: 1047-54, 1999). These authors examined 23 matchedsets of colorectal carcinoma and normal colon mucosa, and found asignificant increase in calgranulin A and B expression in malignanttissues of 70% of donors.

Nucleoside diphosphate kinase A (nm23, SwissProt Accession NumberP15531) is widely regarded as a tumour marker in a variety of cancers,but is the subject of some controversy. In certain tumours such asmetastatic ovarian carcinoma (Viel et al, Cancer Res 55: 2645-2650,1995), malignant melanoma (Florenes et al, Cancer Res 52: 6088-91,1992), hepatocellular carcinoma (Kodera et al, Cancer 73: 259-65, 1994)and prostate cancer (Fishman et al. J Urol. 152: 202-7. 1994) low levelsof expression of nm23 correlate with a highly metastatic phenotype,suggesting a role for the protein in inhibiting the process ofmetastasis. However, among other tumour types, nm23 expression has noapparent relationship to metastatic potential, and may even correlatedirectly with severity in some of these cancers. For example, a 2-foldincrease in nm23 expression is observed in advanced stages of thyroidcarcinoma, suggesting a direct correlation of nm23 expression with rapidcell proliferation in thyroid cancer (Zou et al, Br J Cancer 68: 385-8,1993). Results in colorectal tumours are confusing and contradictory;some researchers report no significant correlation between nm23expression and colorectal tumour histology, serosal invasion, lymphaticinvasion, venous invasion, or lymph node metastasis (Yamaguchi et al, BrJ Cancer 68: 1020-4, 1993) whilst others find that nm23 expressionincreases with local colorectal tumour severity, and reaches even higherlevels in liver metastases (Zeng et al, Br J Cancer 70: 1025-30, 1994).

Over-expression of prohibitin (SwissProt Accession Number P35232) hasbeen reported in tumours from a variety of tissue sources, includingcolon (Coates et al, Exp Cell Res 265: 262-73, 2001) as well as inbreast cancer cell lines (Williams et al, Electrophoresis 19: 333-43,1998).

EXAMPLE 2 Discussion of Novel Markers

TABLE 2 shows 11 novel markers not detectable in normal tissue controls,but found in tumour samples analysed:

Name: hnRNP-K

hnRNP-K is a member of the poly(C) binding proteins (PCBPs), which areinvolved in mRNA stabilization, translational activation, andtranslational silencing. It binds tightly to poly(C) sequences, and islikely to play a role in the nuclear metabolism of hnRNAs, particularlyfor pre-mRNAs that contain cytidine-rich sequences. It is known to beupregulated in SV-40 transformed human keratinocytes (Dejgaard et al, JMol Biol 236: 33-48, 1994), and to be present at higher levels insamples from grade III human breast cancer than in samples from grade IIcancer (Mandal et al, J Biol Chem 276: 9699-704, 2001). Over-expressionof the related protein hnRNP A2/B1(SwissProt P22626) is associated withtumours, for example in the lung (Mulshine et al, Clin Chest Med. 23:37-48, 2002) and pancreas (Yan-Sanders et al, Cancer Lett 183: 215-20,2002), but hnRNP-K has not previously been recognised as a marker ofcolorectal cancer. U.S. Pat. No. 6,358,683 discloses the elevatedexpression of hnRNP-K in breast cancer cells and diagnosis of breastcancer from patient blood samples by assaying hnRNP-K amongst othermarkers.

As shown in Table 2, this protein was not detectable in normal tissuecontrols, but was found in 14 of the 16 colorectal tumour samplesanalysed. The amino acid sequence of this protein, identified as hnRNP-Kby comparison of the experimentally-derived tryptic peptide fingerprintwith entries in the MS-Fit database, is referred to herein as SEQ IDNO:1. hnRNP-K was detected in the tumour of six out of the eightindividuals in the good survival cohort compared with all eight of theindividuals in the poor survival cohort.

Name: HMG-1

HMG-1 (“HMGB1” in the new nomenclature, also called amphoterin) is anuclear architectural chromatin-binding factor that bends DNA andpromotes protein assembly on specific recognition sequences. However,HMG-1 is also secreted by activated monocytes and macrophages, and ispassively released by necrotic or damaged cells into their environment,where it binds with high affinity to RAGE (the receptor for advancedglycation end products) and is a potent mediator of inflammation andimmune response. In apoptotic cells generalized underacetylation ofhistone prevents the release of HMGB1 even after partial autolysis, andthus fail to promote inflammation even if not cleared promptly byphagocytic cells. In this way apoptotic cells are prevented fromgenerating the signal that is broadcast by cells damaged or killed bytrauma (Scaffidi et al, Nature 418: 191-5, 2002). Overexpression ofHMGB1 induced by steroid hormones protects chromatin/platin adducts fromthe nuclear DNA repair apparatus (He et al; PNAS 97: 5768-72, 2000).HMGB1 is found in many cell types and described as a ubiquitous nuclearprotein present at a copy number of ˜10⁶ per typical mammalian cell, butits expression may be associated with a dividing, DNA-replicatingphenotype. As the study disclosed herein involves an enrichment ofpatient samples for cytoplasmic proteins we might expect not to seeHMGB1 in normal samples, and it may be that in tumour cells HMGB1 isreleased from nuclear sequestration and is detected by proteomicexamination of the cytoplasm. The related HMGA proteins (e.g. HMGI(Y))are known tumour antigens; HMGI(Y) protein has been shown to beoverexpressed in various human malignancies, including colon, prostateand thyroid carcinomas (Chiappetta et al, Int. J. Can 91: 147-51, 2001).HMGB1 has not previously been reported as a tumour antigen, and its usein diagnosis of cancers is not described in the patent literature.Modulation of HMG1's interaction with the RAGE receptor, which is knownto stimulate cell mobility and replication and to trigger inflammatoryresponses, is the subject of several patent applications (e.g. WO0047104, WO 02070473, WO 02069965, WO 0192210).

As shown in Table 2, this protein was not detectable in normal tissuecontrols, but was found in 14 of the 16 colorectal tumour samplesanalysed. The amino acid sequence of this protein, identified as HMG-1by comparison of the experimentally-derived tryptic peptide fingerprintwith entries in the MS-Fit database, is referred to herein as SEQ IDNO:2. HMG-1 was detected in the tumours of seven of the eightindividuals in each of the good survival and poor survival cohorts.

Name: Proteasome Subunit Alpha Type 1

Proteasome subunit alpha type 1 (also “proteasome component C2”) is acomponent of the 26S proteasome, a large multicatalytic protease complex(reviewed by Naujokat & Hoffmann, Lab Invest 82: 965-80, 2002, and Coux,Prog Mol Subcell Biol. 29: 85-107, 2002). This complex, which is foundin the cytoplasm and nucleus of all eukaryotic cells, is the terminalstep in the ubiquitin-protease mechanism, which regulates basic cellularprocesses through targeted degradation of regulatory proteins such asthose governing the cell cycle. The 26S proteasome complex is composedof the barrel-shaped 20S catalytic core unit capped at each end by the19S regulatory complex. The non-catalytic alpha subunits form the outersurface of the 26S barrel, and mediate substrate translocation into thecentral cavity and interaction between the 20S and 19S subunits. Whilstderangement of proteasome function is a known feature of certaindiseases, including cancer and neurodegenerative conditions, andinhibition of proteasome function is a therapeutic goal of cancerresearch (see for example Shah et al, Surgery 131: 595-600, 2002),up-regulation of expression of subunit alpha type 1 has not previouslybeen observed in cancer. US5843715 teaches the use ofgenetically-engineered variant proteasome subunits to direct antigenprocessing and presentation towards desired antigens (e.g. tumourantigens, Alzheimer's proteins etc) for therapeutic benefit.

As shown in Table 2, this protein was not detectable in normal tissuecontrols, but was found in 13 of the 16 colorectal tumour samplesanalysed. The amino acid sequence of this protein, identified asproteasome subunit alpha type 1 by comparison of theexperimentally-derived tryptic peptide fingerprint with entries in theMS-Fit database, is referred to herein as SEQ ID NO:3. Proteasomesubunit alpha type 1 was detected in the tumours of six of the eightindividuals in the good survival cohort and seven of the eightindividuals in the poor survival cohort.

Name: Bifunctional Purine Biosynthesis Protein

The human purH gene encodes this 591-amino acid bifunctional proteinwhich exhibits the final two activities of the purine nucleotidebiosynthetic pathway, AICARFT and IMPCH, located within the C-terminaland N-terminal regions, respectively (Rayl et al., J Biol Chem 271:2225-33, 1996; Sugita et al, J Biochem (Tokyo) 122: 309-13 1997). Aswith another enzymatic activity earlier in the pathway, glycinamideribonucleotide formyltransferase (GARFT), it requires a reduced folatecofactor, 10-formyltetrahydrofolate. AICARFT inhibition is thought to bethe origin of the anti-purine effects of anti-folates such asmethotrexate whose primary target is dihydrofolate reductase (Budzik etal, Life Sci 66: 2297-307, 2000). Due to the central role played by thispathway in the synthesis of nucleotides for DNA replication, itscomponent enzymes are of interest as targets for chemotherapy (see forexample Greasley et al, Nat Struct Biol 8: 402-6). Inhibitors of GARFTare currently in clinical trials as anti-neoplastic agents (e.g. TularikInc.'s T64/lometrexol, Eli Lilly's LY309887, Agouron Pharmaceuticals'AG2037). WO0056924 discloses an association between biallelic markers ofa PURH gene and cancer, particularly prostate cancer, and provides meansto determine the predisposition of individuals to cancer as well asmeans for the diagnosis of cancer and for the prognosis/detection of aneventual treatment response to agents acting on cancer. WO9413295 andWO0013688 disclose inhibitors of GARFT and AICARFT, and their use asantiproliferative agents.

As shown in Table 2, this protein was not detectable in normal tissuecontrols, but was found in 12 of the 16 colorectal tumour samplesanalysed. The amino acid sequence of this protein, identified asbifunctional purine biosynthesis protein by comparison of theexperimentally-derived tryptic peptide fingerprint with entries in theMS-Fit database, is referred to herein as SEQ ID NO:4. Bifunctionalpurine biosynthesis protein was detected in the tumours of five of theeight individuals in the good survival cohort compared with seven of theeight individuals in the poor survival cohort.

Name: STI-1

The molecular chaperone Hsp90plays an essential role in the folding andactivation of a set of client proteins involved in cell cycleregulation, signal transduction, and responsiveness to steroid hormone,in a manner dependent on its own endogenous ATPase activity (reviewed byPearl & Prodromou, Curr Opin Struct Biol 10: 46-51, 2000). Fromexperiments in yeast it is known that for some client proteins theco-chaperone stress-inducible protein 1 (STI1, also called Hsp70/Hsp90organizing protein (Hop) or p60) acts as a scaffold for assembly of theHsp70/Hsp90 chaperone heterocomplex, recruiting Hsp70 and the boundclient to Hsp90 and sterically inhibiting Hsp90 ATPase activity duringassembly (Johnson et al, J Biol Chem 273: 3679-86, 1998; Richter et al,J Biol Chem. Jan. 13, 2003 [e-publication ahead of print]). Thephosphorylation of murine STI1 by casein kinase II (CKII) at S189 and bycdc2 kinase (p34cdc2) at T198 has been implicated as a potential cellcycle checkpoint (Longshaw et al, Biol Chem 381: 1133-8, 2000). STI1 hasalso been observed to bind TCP-1, subunits beta and epsilon of whichwere also identified as cancer markers in the present study, stimulatingits nucleotide exchange activity (Gebauer et al, J Biol Chem 273:29475-80, 1998).

As shown in Table 2, this protein was not detectable in normal tissuecontrols, but was found in 12 of the 16 colorectal tumour samplesanalysed. The amino acid sequence of this protein, identified as STI1 bycomparison of the experimentally-derived tryptic peptide fingerprintwith entries in the MS-Fit database, is referred to herein as SEQ IDNO:5. STI1 was detected in the tumours of five of the eight individualsin the good survival cohort and seven of the eight individuals in thepoor survival cohort.

Name: Annexin IV

Annexin IV (also called annexin A4) is a member of the annexin family ofCa2+- and phospholipid-binding proteins, which have poorly definedfunctions in membrane-related events along exocytotic and endocytoticpathways. Annexin IV is threonine phosphorylated by protein kinase C(Kaetzel et al, Biochemistry 40: 4192-9, 2001), binds to surfactantprotein A in a Ca²+-dependent manner (Sohma et al, Biochem. J. 312:175-81, 1995) and may bind to glycosylphosphatidylinositol-anchoredglycoprotein GP-2, a major component of the zymogen granule membrane(Tsujii-Hayashi et al, J Biol Chem 277: 47493-9, 2002). IHC analysis ina broad variety of human tissues indicates that annexin IV is almostexclusively found in epithelial cells (Dreier et al, Histochem Cell Biol110: 137-48, 1998). The over-expression of annexin IV in C6 cells bytransfection with annexin IV-DNA induces activation of NFkappaB (Ohkawaet al, Biochim Biophys Acta 1588: 217, 2002), while the Fas-induced celldeath of Jurkat T-lymphocytes is accompanied by translocation of annexinIV from the nucleus to the cytosol (Gerner et al, J Biol Chem 275:39018-26, 2000). The protein also plays a role in the paclitaxelresistant phenotype of the H460/T800 cell line and is among the earliestproteins induced in cells in response to cytotoxic stress such asantimitotic drug treatment (Han et al, Br J Cancer 83: 83-8, 2000).Elevated levels of annexin IV have been demonstrated by specificdouble-antibody radioimmunoassay in the sera of 47.3% (35 of 74)cervical cancer patients and 41.9% (18 of 43) endometrial cancerpatients (Gocze et al, Strahlenther Onkol 167: 538-44, 1991). Therelated protein annexin II appears to be overexpressed in advancedcolorectal carcinoma and may be related to the progression andmetastatic spread of the disease (Emoto et al, Cancer 92: 1419-26, 2001)but annexin IV has not previously been identified as a marker ofcolorectal tumours. WO0111372 discusses the diagnosis of cancers bymeans of detecting increased expression of annexin proteins inbiological samples, but provides examples of annexins I and II only,whilst WO0012547 discusses use of annexin V as a marker in the diagnosisof cancer. U.S. Pat. No. 5,316,915 describes the production of anantibody capture assay to detect antibodies against annexins (includingannexin IV) within human body fluids.

As shown in Table 2, this protein was not detectable in normal tissuecontrols, but was found in 11 of the 15 colorectal tumour samplesanalysed. The amino acid sequence of this protein, identified as annexinIV by comparison of the experimentally-derived tryptic peptidefingerprint with entries in the MS-Fit database, is referred to hereinas SEQ ID NO:6. Annexin IV was detected in the tumours of four of theeight individuals in the good survival cohort compared with seven of theseven individuals in the poor survival cohort, showing that thisparticular protein constitutes a useful prognostic indicator in certaincancers such as colorectal carcinoma.

Name: 60 kDa Heat Shock Protein

60 kDa heat shock protein (hsp60) is abundant in a variety of mammaliancells under normal conditions (Welch et al, Physiol. Rev. 72: 1063-81,1992), where its major functions are protein chaperoning and proteinfolding (reviewed by Bukau & Horwich, Cell 92: 351-66, 1998). Bothprocesses are co-regulated by hsp10. hsp60 has been shown to bind adiverse range of cellular protein components; recently identifiedbinding partners include the calcineurin B regulatory subunit of theCa²⁺/calmodulin-dependent protein phosphatase calcineurin (Li &Handschumacher, Biochim Biophys Acta 1599: 72-81, 2002), the humanhepatitis B virus polymerase (Park & Jung, J. Virol. 75: 6962-8, 2001),integrin alpha 3 beta 1 (Barazi et al, Cancer Res 62: 1541-8, 2002), theinfectious prion protein PrP (Stockel & Hartl, J Mol Biol 313: 861-7,2001) and a receptor molecule on macrophages found to be distinct fromreceptors for other members of the heat shock protein family (Habich etal, J Immunol 168: 569-76, 2002).Whereas aberrant expression of hsp60has been associated with autoimmune disease, hsp6o has a role togetherwith hsp7o in antigen presentation in malignant diseases (Multhoff etal, Int. J. Cancer 61: 272-279, 1995), with enhanced hsp60 expressionreported in myeloid leukaemia (Chant et al, Br. J. Haematol., 90: 163-8,1995), breast carcinoma (Franzen et al, Electrophoresis, 18: 582-7,1997; Bini et al, Electrophoresis 18: 2832-41, 1997) and prostatecancers (Cornford et al, Cancer Res 60: 7099-105, 2000). Two proteins of40 kDa and 47 kDa have been detected in colorectal tumours and not innormal tissue via immunoblotting with an antibody to hsp60(Otaka et al,J Clin Gastroenterol 21: 224-9, 1995), but up-regulation of hsp60expression in colorectal tumours has not previously been definitivelyobserved. U.S. Pat. No. 5,434,046 discusses prognosis of ovarian cancertreated with cis-platin by measurement of Hsp60 expression.

As shown in Table 2, this protein was not detectable in normal tissuecontrols, but was found in 11 of the 16 colorectal tumour samplesanalysed. The amino acid sequence of this protein, identified as 60 kDaheat shock protein by comparison of the experimentally-derived trypticpeptide fingerprint with entries in the MS-Fit database, is referred toherein as SEQ ID NO:7. 60 kDa heat shock protein was detected in thetumours of four of the eight individuals in the good survival cohortcompared with seven of the eight individuals in the poor survivalcohort, showing that this particular protein constitutes a usefulprognostic indicator in certain cancers such as colorectal carcinoma.

Name: T Complex Protein 1 Beta Subunit

The TCP-1 ring complex (TRiC; also called CCT, for chaperonin containingTCP-1) is a large (approximately 900 kDa) ring-shaped multisubunitcomplex consisting of eight different, yet homologous, subunits rangingbetween 50 and 60 kDa, which include the TCP-1 beta species (Frydman etal, EMBO J. 11: 4767-78, 1992; Gao et al, Cell 69:1043-50, 1992; Lewiset al, Nature 358: 249-252, 1992). TCP-1 mediates protein folding of anas yet poorly defined physiological substrate spectrum in the eukaryoticcytosol, binding client proteins within its central cavity and inducingtheir folding by an ATP-dependent mechanism. Genetic and biochemicaldata show that it is required for the folding of the cytoskeletalproteins actin and tubulin, and TCP-1 was originally proposed to be achaperone specialized for the folding of these proteins (Lewis et al, J.Cell Biol. 132: 1-4, 1996). However, recent experiments suggest abroader substrate spectrum of distinct proteins that require TCP-1 forproper folding in vivo, including the Von Hippel-Lindau tumoursuppressor protein and cyclin E (Dunn et al, J Struct Biol 135: 176-84,2001). Analysis of auto-antibodies in sera from astrocytoma patientsidentified TCP-1 as a potential preferentially-expressed antigen in suchtumours (Schmits R et al, Int J Cancer 98: 73-7, 2002), and expressionof the TCP-1 subunit has been linked with the S to G2/M phase transitionof the cell cycle (Dittmar et al, Cell Biol Int 21: 383-91, 1997).

As shown in Table 2, this protein was not detectable in normal tissuecontrols, but was found in 11 of the 16 colorectal tumour samplesanalysed. The amino acid sequence of this protein, identified as Tcomplex protein 1 beta subunit by comparison of theexperimentally-derived tryptic peptide fingerprint with entries in theMS-Fit database, is referred to herein as SEQ ID NO:8. T complex protein1 beta subunit was detected in the tumours of five of the eightindividuals in the good survival cohort and six of the eight individualsin the poor survival cohort.

Name: T Complex Protein 1 Epsilon Subunit

The TCP-1 ring complex (TRiC; also called CCT, for chaperonin containingTCP-1) is a large (approximately 900 kDa) ring-shaped multisubunitcomplex consisting of eight different, yet homologous, subunits rangingbetween 50 and 60 kDa, which include the TCP-1 epsilon species (Frydmanet al, EMBO J. 11: 4767-78, 1992; Gao et al, Cell 69:1043-50, 1992;Lewis et al, Nature 358: 249-252, 1992). TCP-1 mediates protein foldingof an as yet poorly defined physiological substrate spectrum in theeukaryotic cytosol, binding client proteins within its central cavityand inducing their folding by an ATP-dependent mechanism. Genetic andbiochemical data show that it is required for the folding of thecytoskeletal proteins actin and tubulin, and TCP-1 was originallyproposed to be a chaperone specialized for the folding of these proteins(Lewis et al, J. Cell Biol. 132: 1-4, 1996). However, recent experimentssuggest a broader substrate spectrum of distinct proteins that requireTCP-1 for proper folding in vivo, including the Von Hippel-Lindau tumoursuppressor protein and cyclin E (Dunn et al, J Struct Biol 135: 176-84,2001). Epstein-Barr virus-encoded nuclear protein EBNA-3 is known tointeract with the TCP-1 epsilon-subunit (Kashuba et al, J Hum Virol. 2:33-7, 1999). Analysis of auto-antibodies in sera from astrocytomapatients identified TCP-1 as a potential preferentially-expressedantigen in such tumours (Schmits R et al, Int J Cancer 98: 73-7, 2002),and expression of the TCP-1 subunit has been linked with the S to G2/Mphase transition of the cell cycle (Dittmar et al, Cell Biol Int 21:383-91, 1997).

As shown in Table 2, this protein was not detectable in normal tissuecontrols, but was found in 11 of the 16 colorectal tumour samplesanalysed. The amino acid sequence of this protein, identified as Tcomplex protein 1 epsilon subunit by comparison of theexperimentally-derived tryptic peptide fingerprint with entries in theMS-Fit database, is referred to herein as SEQ ID NO:9. T complex protein1 epsilon subunit was detected in the tumour of only four of the eightindividuals in the good survival cohort compared with seven of the eightindividuals in the poor survival cohort, showing that this particularprotein constitutes a useful prognostic indicator in certain cancerssuch as colorectal carcinoma.

Name: Mortalin

Human mortalin (also called stress-70 protein) is a member of the hsp 70family of proteins initially identified by virtue of its associationwith a cellular mortal phenotype. The subcellular localisation ofmortalin is distinct in normal and immortalised cells, with cytosolicand perinuclear distribution patterns distinguishing the mortalphenotype from the immortal, respectively. Consistently, the cytosolicmortalin is seen to have a senescence-inducing function in contrast tothe perinuclear mortalin which has no detectable effect on cellularphenotype (Wadhwa et al, Histol Histopathol 17: 1173-7, 2002; Kaul etal, Exp Gerontol 37: 1157-64, 2002). The human mortalin gene, HSPA9, hasbeen localized to chromosome 5, band q31, a region that is frequentlydeleted in myeloid leukaemias and myelodysplasia, making it a candidatetumour suppressor gene, and has presumed functions in the stressresponse, intracellular trafficking, antigen processing, control of cellproliferation, differentiation and tumourigenesis. Transfection ofmurine 3T3 cells with human mortalin cDNA results in their malignanttransformation (Wadhwa et al, J Biol Chem 268: 6615-21, 1993), as doestransfection with the murine homologue mot-2 (Kaul et al, Oncogene 17:907-11, 1998), which is known to bind and inactivate the tumoursuppressor protein p53 (Wadhwa et al, J. Biol. Chem 273: 29586-91,1998). A number of binding partners for mortalin have confirmed,including glucose regulated protein 94 (Takano et al, Biochem J. 357:393-8, 2001), fibroblast growth factor 1 (Mizukoshi et al, Biochem J.343: 461-6, 1999) and the interleukin 1 receptor type 1 (Sacht et al,Biofactors 9: 49-60, 1999). Proteomic analysis of human breast ductalcarcinoma and histologically normal tissue has identified mortalin ashighly expressed in all carcinoma specimens, and less intense andoccasionally undetectable in normal tissue (Bini et al, Electrophoresis18: 2832-41, 1997). Mortalin expression is also elevated in a variety ofneurological tumours, including low-grade astrocytoma, anaplasticastrocytoma, glioblastoma, meningiomas, neurinomas, pituitary adenomas,and metastases thereof (Takano et al, Exp Cell Res 237: 38-45, 1997).W00144807 describes a screening system for drugs that disruptinteraction of p53 with mortalin. U.S. Pat. No. 5,627,0394 discussescharacterisation of intracellular mortalin expression with ananti-mortalin antibody and comparison with the “complementation group”of immortalised cells, which is described as an indicator of cellularmortality/immortality phenotype.

As shown in Table 2, this protein was not detectable in normal tissuecontrols, but was found in 9 of the 16 colorectal tumour samplesanalysed. The amino acid sequence of this protein, identified asmortalin by comparison of the experimentally-derived tryptic peptidefingerprint with entries in the MS-Fit database, is referred to hereinas SEQ ID NO:10. Mortalin was detected in the tumour of only three ofthe eight individuals in the good survival cohort compared with six ofthe eight individuals in the poor survival cohort, showing that thisparticular protein constitutes a useful prognostic indicator in certaincancers such as colorectal carcinoma.

Name: TER-ATPase

TER ATPase, also called valosin-containing protein (VCP), is themammalian homologue of the Saccharomyces cerevisiae cell cycle controlprotein cdc48p (reviewed by Wojcik, Trends Cell Biol 12: 212, 2002). Itis a homohexameric protein exhibiting a nuclear and cytoplasmicdistribution, which has multiple biological functions and has been shownin murine cells to be tyrosine phosphorylated in response to T cellantigen receptor activation, which may provide a link between TCR ligandbinding and cell cycle control (Egerton et al, EMBO J 11: 3533-40,1992). TER ATPase acts as a chaperone in membrane fusions involved inthe transfer of transition vesicles from the transitional endoplasmicreticulum to the Golgi apparatus. It also physically targetsubiquitinated nuclear factor kappaB inhibitor to the proteasome fordegradation (Dai et al, J Biol Chem 273: 3562-73, 1998). Loss of TERATPase function results in an inhibition of Ub-Pr-mediated degradationand an accumulation of ubiquitinated proteins, suggesting a role in thedegradation of multiple Ub-Pr pathway substrates (Dai & Li, Nat CellBiol 3: 740-4, 2001). TER ATPase is reported to bind residues 303-625 ofthe BRCA1 protein via its N-terminal region, and may thereby participateas an ATP transporter in DNA damage-repair functions (Zhang et al, DNACell Biol 19: 253-63, 2000). Stimulation of TER ATPase-transfected Dunncells (a murine osteosarcoma cell line) with TNFalpha induced persistentactivation of NFkappaB via enhanced cytoplasmic degradation ofp-IkappaBalpha, a reduced rate of apoptosis and increased metastaticpotential when used to inoculate male C3H mice (Asai et al, Jpn J CancerRes 93: 296-304, 2002). Hepatocellular carcinoma (HCC) patients who hadmore TER ATPase in their tumours than in normal endothelial tissueshowed a higher rate of portal vein invasion in the tumour and poorerdisease-free and overall survival than patients in whose tumour cellsthe staining intensity was weaker than in normal tissue, making TERATPase a prognostic indicator in patients with HCC (Yamamoto et al, JClin Oncol 21: 447-52, 2003). W00216938 discusses a method of screeningfor compounds that treat neurodegenerative diseases by inhibitingbinding of TER ATPase to an abnormal protein substrate.

As shown in Table 2, this protein was not detectable in normal tissuecontrols, but was found in 7 of the 16 colorectal tumour samplesanalysed. The amino acid sequence of this protein, identified as TERATPase by comparison of the experimentally-derived tryptic peptidefingerprint with entries in the MS-Fit database, is referred to hereinas SEQ ID NO:11. TER ATPase was detected in the tumour of only one ofthe eight individuals in the good survival cohort compared with six ofthe eight individuals in the poor survival cohort, showing that thisparticular protein constitutes a useful prognostic indicator in certaincancers such as colorectal carcinoma.

TABLE 1 Differentially expressed proteins identified in the presentstudy and already known to be upregulated in colorectal cancerAbbreviated SwissProt frequency of name Accession No. full nameup-regulation calgranulin P05109 S100 calcium binding 10/13 (77%) Aprotein A8 calgranulin P06702 S100 calcium binding 12/16 (75%) B proteinA9 nm-23 P15531 nucleoside diphosphate 12/16 (75%) kinase A — P35232prohibitin  9/16 (56%)

TABLE 2 Proteins detectable in colorectal cancer samples but not normalcolon tissue controls: novel findings of the present study SwissProtAbbreviated Accession frequency of name No. full name up-regulationhnRNP K Q07244 heterogeneous nuclear 14/16 (88%) riboprotein K HMGB1P09429 high mobility group protein 1 14/16 (88%) (amphoterin) — P25786proteasome subunit alpha type 1 13/16 (81%) PURH P31939 bifunctionalpurine 12/16 (75%) biosynthesis protein STI1 P31948 stress-inducedphosphoprotein 1 12/16 (75%) — P09525 annexin IV (annexin A4) 11/15(73%) Hsp60 P10809 60 kDa heat shock protein 11/16 (69%) TCP-Iβ P78371T-complex protein 1, beta 11/16 (69%) subunit TCP-1ε P48643 T-complexprotein 1, epsilon 11/69 (69%) subunit mortalin P38646 mitochondrialstress-70 protein  9/16 (56%) TER ATPase P55072 transitional endoplasmic7/16 (44) reticulum ATPase

TABLE 3 Clinicopathological characteristics of the cases used forproteome analysis. All the cases were Dukes C colorectal cancers.characteristics Sex Male: n = 8 Female: n = 8 Age <55 years: n = 6 ≧55years: n = 10 Site Proximal: n = 8 Distal: n = 8 Tumour differentiationWell: n = 0 Moderate: n = 15 Poor: n = 1

TABLE 4 full name example ligands heterogeneous cytidine-richpolyribonucleotides nuclear riboprotein K high mobility group Chromatinprotein 1 (amphoterin) proteasome subunit other components of the 26Sproteasome, alpha type 1 including the 20S and 19S subunits bifunctionalpurine the purine synthesis intermediates AICAR biosynthesis protein(aminoimidazole carboxamide ribonucleotide) and FAICAR(formylaminoimidazole carboxamide ribonucleotide) stress-induced 70 kDaheat shock protein, 90 kDa heat phosphoprotein 1 shock protein, case inkinase II, cdc2 kinase or T complex protein 1 annexin IV protein kinaseC, surfactant protein A or (annexin A4)glycosylphosphatidylinositol-anchored glycoprotein GP-2. 60 kDa heatshock calcineurin B, the human hepatitis B protein virus polymerase,integrin alpha 3 beta 1 or the infectious prion protein PrP T-complexprotein 1, actin, tubulin, the Von Hippel-Lindau beta subunit tumoursuppressor protein or cyclin E T-complex protein 1, actin, tubulin, theVon Hippel-Lindau epsilon subunit tumour suppressor protein, cyclin E orthe Epstein-Barr virus-encoded nuclear protein EBNA-3 mitochondrialGlucose regulated protein 94, fibroblast stress-70 growth factor 1 orthe interleukin 1 protein receptor type 1 transitional nuclear factorkappa B inhibitor endoplasmic reticulum ATPase

SEQ ID NO:1 - hnRNP-K METEQPEETF PNTETNGEFG KRPAEDMEEE QAFKRSRNTDEMVELRILLQ SKNAGAVIGK GGKNIKALRT DYNASVSVPD SSGPERILSI SADIETIGEILKKIIPTLEE GLQLPSPTAT SQLPLESDAV ECLNYQHYKG SDFDCELRLL IHQSLAGGIIGVKGAKIKEL RENTQTTIKL FQECCPHSTD RVVLIGGKPD RVVECIKIIL DLISESPIKGRAQPYDPNFY DETYDYGGFT MMFDDRRGRP VGFPMRGRGG FDRMPPGRGG RPMPPSRRDYDDMSPRRGPP PPPPGRGGRG GSRARNLPLP PPPPPRGGDL MAYDRRGRPG DRYDGMVGFSADETWDSAID TWSPSEWQMA YEPQGGSGYD YSYAGGRGSY GDLGGPIITT QVTIPKDLAGSIIGKGGQRI KQIRHESGAS IKIDEPLEGS EDRIITITGT QDQIQNAQYL LQNSVKQYSG KFFSEQ ID NO:2 - HMG-1 GKGDPKKPRG KMSSYAFFVQ TCREEHKKKH PDASVNFSEFSKKCSERWKT MSAKEKGKFE DMAKADKARY EREMKTYIPP KGETKKKFKD PNAPKRPPSAFFLFCSEYRP KIKGEHPGLS IGDVAKKLGE MWNNTAADDK QPYEKKAAKL KEKYEKDIAAYRAKGKPDAA KKGVVKAEKS KKKKEEEEDE EDEEDEEEEE DEEDEDEEED DDDE SEQ IDNO:3 - proteasome subunit alpha type 1 MFRNQYDNDV TVWSPQGRIH QIEYAMEAVKQGSATVGLKS KTHAVLVALK RAQSELAAHQ KKILHVDNHI GISIAGLTAD ARLLCNFMRQECLDSRFVFD RPLPVSRLVS LIGSKTQIPT QRYGRRPYGV GLLIAGYDDM GPHIFQTCPSANYFDCRAMS IGARSQSART YLERHMSEFM ECNLNELVKH GLRALRETLP AEQDLTTKNVSIGIVGKDLE FTIYDDDDVS PFLEGLEERP QRKAQPAQPA DEPAEKADEP MEH SEQ ID NO:4 -bifunctional purine biosynthesis protein MAPGQLALFS VSDKTGLVEFARNLTALGLN LVASGGTAKA LRDAGLAVRD VSELTGFPEM LGGRVKTLHP AVHAGILARNIPEDNADMAR LDFNLIRVVA CNLYPFVKTV ASPGVTVEEA VEQIDIGGVT LLRAAAKNHARVTVVCEPED YVVVSTEMQS SESKDTSLET RRQLALKAFT HTAQYDEAIS DYFRKQYSKGVSQMPLRYGM NPHQTPAQLY TLQPKLPITV LNGAPGFINL CDALNAWQLV KELKEALGIPAAASFKHVSP AGAAVGIPLS EDEAKVCMVY DLYKTLTPIS AAYARARGAD RMSSFGDFVALSDVCDVPTA KIISREVSDG IIAPGYEEEA LTILSKKKNG NYCVLQMDQS YKPDENEVRTLFGLHLSQKR NNGVVDKSLF SNVVTKNKDL PESALRDLIV ATIAVKYTQS NSVCYAKNGQVIGIGAGQQS RIHCTRLAGD KANYWWLRHH PQVLSMKFKT GVKRAEISNA IDQYVTGTIGEDEDLIKWKA LFEEVPELLT EAEKKEWVEK LTEVSISSDA FFPFRDNVDR AKRSGVAYIAAPSGSAADKV VIEACDELGI ILAHTNLRLF HH SEQ ID NO:5 - STI1 MEQVNELKEKGNKALSVGNI DDALQCYSEA IKLDPHNHVL YSNRSAAYAK KGDYQKAYED GCKTVDLKPDWGKGYSRKAA ALEFLNRFEE AKRTYEEGLK HEANNPQLKE GLQNMEARLA ERKFMNPFNMPNLYQKLESD PRTRTLLSDP TYRELIEQLR NKPSDLGTKL QDPRIMTTLS VLLGVDLGSMDEEEEIATPP PPPPPKKETK PEPMEEDLPE NKKQALKEKE LGNDAYKKKD FDTALKHYDKAKELDPTNMT YITNQAAVYF EKGDYNKCRE LCEKAIEVGR ENREDYRQIA KAYARIGNSYFKEEKYKDAI HFYNKSLAEH RTPDVLKKCQ QAEKILKEQE RLAYINPDLA LEEKNKGNECFQKGDYPQAM KHYTEAIKRN PKDAKLYSNR AACYTKLLEF QLALKDCEEC IQLEPTFIKGYTRKAAALEA MKDYTKAMDV YQKALDLDSS CKEAADGYQR CMMAQYNRHD SPEDVKRRAMADPEVQQIMS DPAMRLILEQ MQKDPQALSE HLKNPVIAQK IQKLMDVGLI AIR SEQ ID NO:6 -annexin IV ATKGGTVKAA SGFNAMEDAQ TLRKAMKGLG TDEDAIISVL AYRNTAQRQEIRTAYKSTIG RDLIDDLKSE LSGNFEQVIV GMMTPTVLYD VQELRRAMKG AGTDEGCLIEILASRTPEEI RRISQTYQQQ YGRSLEDDIR SDTSFMFQRV LVSLSAGGRD EGNYLDDALVRQDAQDLYEA GEKKWGTDEV KFLTVLCSRN RNHLLHVFDE YKRISQKDIE QSIKSETSGSFEDALLAIVK CMRNKSAYFA EKLYKSMKGL GTDDNTLIRV MVSRAEIDML DIRAHFKRLYGKSLYSFIKG DTSGDYRKVL LVLCGGDD SEQ ID NO:7 - 60 kDa heat shock proteinMLRLPTVFRQ MRPVSRVLAP HLTRAYAKDV KFGADARALM LQGVDLLADA VAVTMGPKGRTVIIEQSWGS PKVTKDGVTV AKSIDLKDKY KNIGAKLVQD VANNTNEEAG DGTTTATVLARSIAKEGFEK ISKGANPVEI RRGVMLAVDA VIAELKKQSK PVTTPEEIAQ VATISANGDKEIGNIISDAM KKVGRKGVIT VKDGKTLNDE LEIIEGMKFD RGYISPYFIN TSKGQKCEFQDAYVLLSEKK ISSIQSIVPA LEIANAHRKP LVIIAEDVDG EALSTLVLNR LKVGLQVVAVKAPGFGDNRK NQLKDMAIAT GGAVFGEEGL TLNLEDVQPH DLGKVGEVIV TKDDAMLLKGKGDKAQIEKR IQEIIEQLDV TTSEYEKEKL NERLAKLSDG VAVLKVGGTS DVEVNEKKDRVTDALNATRA AVEEGIVLGG GCALLRCIPA LDSLTPANED QKIGIEIIKR TLKIPAMTIAKNAGVEGSLI VEKIMQSSSE VGYDAMAGDF VNMVEKGIID PTKVVRTALL DAAGVASLLTTAEVVVTEIP KEEKDPGMGA MGGMGGGMGG GMF SEQ ID NO:8 - T complex protein 1beta subunit MASLSLAPVN IFKAGADEER AETARLTSFI GAIAIGDLVK STLGPKGMDKILLSSGRDAS LMVTNDGATI LKNIGVDNPA AKVLVDMSRV QDDEVGDGTT SVTVLAAELLREAESLIAKK IHPQTIIAGW REATKAAREA LLSSAVDHGS DEVKFRQDLM NIAGTTLSSKLLTHHKDHFT KLAVEAVLRL KGSGNLEAIH IIKKLGGSLA DSYLDEGFLL DKKIGVNQPKRIENAKILIA NTGMDTDKIK IFGSRVRVDS TAKVAEIEHA EKEKMKEKVE RILKHGINCFINRQLIYNYP EQLFGAAGVM AIEHADFAGV ERLALVTGGE IASTFDHPEL VKLGSCKLIEEVMIGEDKLI HFSGVALGEA CTIVLRGATQ QILDEAERSL HDALCVLAQT VKDSRTVYGGGCSEMLMAHA VTQLANRTPG KEAVAMESYA KALRMLPTII ADNAGYDSAD LVAQLRAAHSEGNTTAGLDM REGTIGDMAI LGITESFQVK RQVLLSAAEA AEVILRVDNI IKAAPRKRVP DHHPCSEQ ID NO:9 - T complex protein 1 epsilon subunit MASMGTLAFD EYGRPFLIIKDQDRKSRLMG LEALKSHIMA AKAVANTMRT SLGPNGLDKM MVDKDGDVTV TNDGATILSMMDVDHQIAKL MVELSKSQDD EIGDGTTGVV VLAGALLEEA EQLLDRGIHP IRIADGYEQAARVAIEHLDK ISDSVLVDIK DTEPLIQTAK TTLGSKVVNS CHRQMAEIAV NAVLTVADMERRDVDFELIK VEGKVGGRLE DTKLIKGVIV DKDFSHPQMP KKVEDAKIAI LTCPFEPPKPKTKHKLDVTS VEDYKALQKY EKEKFEEMIQ QIKETGANLA ICQWGFDDEA NHLLLQNNLPAVRWVGGPEI ELIAIATGGR IVPRFSELTA EKLGFAGLVQ EISFGTTKDK MLVIEQCKNSRAVTIFIRGG NKMIIEEAKR SLHDALCVIR NLIRDNRVVY GGGAAEISCA LAVSQEADKCPTLEQYAMRA FADALEVIPM ALSENSGMNP IQTMTEVRAR QVKEMNPALG IDCLHKGTNDMKQQHVIETL IGKKQQISLA TQMVRMILKI DDIRKPGESE E SEQ ID NO:10 - mortalinMISASRAAAA RLVGAAASRG PTAARHQDSW NGLSHEAFRL VSRRDYASEA IKGAVVGIDLGTTNSCVAVM EGKQAKVLEN AEGARTTPSV VAFTADGERL VGMPAKRQAV TNPNNTFYATKRLIGRRYDD PEVQKDIKNV PFKIVRASNG DAWVEAHGKL YSPSQIGAFV LMKMKETAENYLGHTAKNAV ITVPAYFNDS QRQATKDAGQ ISGLNVLRVI NEPTAAALAY GLDKSEDKVIAVYDLGGGTF DISILEIQKG VFEVKSTNGD TFLGGEDFDQ ALLRHIVKEF KRETGVDLTKDNMALQRVRE AAEKAKCELS SSVQTDINLP YLTMDSSGPK HLNMKLTRAQ FEGIVTDLIRRTIAPCQKAM QDAEVSKSDI GEVILVGGMT RMPKVQQTVQ DLFGRAPSKA VNPDEAVAIGAAIQGGVLAG DVTDVLLLDV TPLSLGIETL GGVFTKLINR NTTIPTKKSQ VFSTAADGQTQVEIKVCQGE REMAGDNKLL GQFTLIGIPP APRGVPQIEV TFDIDANGIV HVSAKDKGTGREQQIVIQSS GGLSKDDIEN MVKNAEKYAE EDRRKKERVE AVNMAEGIIH DTETKMEEFKDQLPADECNK LKEEISKMRE LLARKDSETG ENIRQAASSL QQASLKLFEM AYKKMASEREGSGSSGTGEQ KEDQKEEKQ SEQ ID NO:11 - TER-ATPase MASGADSKGD DLSTAILKQKNRPNRLIVDE AINEDNSVVS LSQPRMDELQ LFRGDTVLLK GKKRREAVCI VLSDDTCSDEKIRMNRVVRN NLRVRLGDVI SIQPCPDVKY GKRIHVLPID DTVEGITGNL FEVYLKPYFLEAYRPIRKGD IFLVRGGMRA VEFKVVETDP SPYCIVAPDT VIHCEGEPIK REDEEESLNEVGYDDIGGCR KQLAQIKEMV ELPLRHPALF KAIGVKPPRG ILLYGPPGTG KTLIARAVANETGAFFFLIN GPEIMSKLAG ESESNLRKAF EEAEKNAPAI IFIDELDAIA PKREKTHGEVERRIVSQLLT LMDGLKQRAH VIVMAATNRP NSIDPALRRF GRFDREVDIG IPDATGRLEILQIHTKNMKL ADDVDLEQVA NETHGHVGAD LAALCSEAAL QAIRKKMDLI DLEDETIDAEVMNSLAVTMD DFRWALSQSN PSALRETVVE VPQVTWEDIG GLEDVKRELQ ELVQYPVEHPDKFLKFGMTP SKGVLFYGPP GCGKTLLAKA IANECQANFI SIKGPELLTM WFGESEANVREIFDKARQAA PCVLFFDELD SIAKARGGNI GDGGGAADRV INQILTEMDG MSTKKNVFIIGATNRPDIID PAILRPGRLD QLIYIPLPDE KSRVAILKAN LRKSPVAKDV DLEFLAKMTNGFSGADLTEI CQRACKLAIR ESIESEIRRE RERQTNPSAM EVEEDDPVPE IRRDHFEEAMRFARRSVSDN DIRKYEMFAQ TLQQSRGFGS FRFPSGNQGG AGPSQGSGGG TGGSVYTEDN DDDLYG

1. An in vitro method of identifying the presence of colorectal cancercells in a test sample of tissue from an individual, said tissuecomprising at least one of colon and rectal tissue, which methodcomprises: (a) determining whether a target protein is over-expressed incells from said test sample compared to expression of the same proteinin a second clinically normal tissue sample comprising at least one ofcolon and rectal tissue from the same individual or a second healthyindividual, said target protein being mitochondrial stress-70 protein ofSEQ ID NO: 10, and (b) correlating over-expression of said targetprotein with the presence of said cancer cells in said test sample,wherein over-expression of said protein indicates the presence of saidcancer cell in said test sample.
 2. A method as claimed in claim 1,wherein the target protein is detected using a recognition agent, saidrecognition agent being an antibody, or a binding moiety of an antibody,which is effective to specifically bind the target protein, and saidrecognition agent is optionally linked to a detectable label.
 3. Amethod as claimed in claim 2 wherein the method comprises (a) obtainingfrom a patient a tissue sample comprising at least one of colon andrectal tissue to be tested for the presence of cancer cells; (b)producing a prepared sample of said tissue in a sample preparationprocess; (c) contacting the prepared sample with said recognition agentthat binds to the target protein; and (d) detecting binding of therecognition agent to the target protein, if present, in the preparedsample.
 4. A method as claimed in claim 2 wherein the recognition agentis an antibody.